The long range goal of this program of research is to clarify the molecular mechanisms by which neurons in the limbic system and hypothalamus integrate endocrine and sensory influences on reproductive function. The proposed project will utilize the anteroventral periventricular nucleus of the hypothalamus (AVPv) as a model system for this research because it is known to play a critical role in mediating hormonal feedback on gonadotropin secretion and receives neurotransmitter-specific afferents from hormone- sensitive regions of the forebrain that relay olfactory information to the hypothalamus. Moreover, it contains sexually dimorphic populations of tyrosine hydroxylase (TH), prodynorphin (PDYN), and proenkephalin (PENK) mRNA-containing neurons that show three distinct patterns of gene expression over the estrous cycle: TH expression is highest during metestrus, but PENK mRNA remains constant, while PDYN mRNA is selectively induced during estrus, indicating that progesterone may play a role in regulating this opioid peptide gene. Because neither the TH nor PDYN gene contains consensus estrogen-regulated enhancer sequences, the observed regulatory patterns are most likely due to indirect mechanisms. The overall objective of the proposed project is to identify molecular events that mediate the hormonal regulation of TH and PDYN in the AVPv by (1) identifying molecules involved in stimulus-transcription coupling that are differentially expressed in TH and PDYN mRNA-containing neurons, and (2) determining if the indirect regulation of TH and PDYN gene expression by estrogen and progesterone is mediated by the nuclear trans-acting factors CREB (cAMP regulated enhancer binding protein), c-fos and c-jun, or by hormone-sensitive, neurotransmitter-specific inputs to these cells. These broad goals will be accomplished by first, using a double in situ hybridization technique to determine if differences in the hormonal regulation of TH and PDYN mRNA levels correspond to differential patterns of co-expression of estrogen and progesterone receptor mRNA in these neurons (Specific Aim 1). Second, in situ and solution hybridization will be used to evaluate possible hormonally induced changes in the expression of CREB, c-fos and c-jun in the AVPv, and to determine whether TH and PDYN mRNA-containing neurons differentially express these trans-acting factors (Specific Aim 2). Third, because the AVPv receives strong SP- immunoreactive inputs from other hormone-sensitive regions, the hormonal regulation of substance P receptor mRNA and possible co-expression in female rats will be evaluated following treatment with substance P agonist or antagonist to determine if these peptidergic afferents trans- synaptically regulate gene expression in the AVPv (Specific Aim 4). The results of these experiments will contribute to our emerging understanding of neuroendocrine regulation of transmitter plasticity within forebrain circuits that mediate key reproductive events, and may provide vital clues about mechanisms that underlie hormone-dependent abnormalities in the neural control of ovulation and fertility.